G protein-coupled P2Y receptors (P2Y-R) for extracellular nucleotides subserve important roles in lung epithelial cell biology and pathophysiology. The long-term goal of our research is to delineate the molecular basis for nucleotide action at airway P2Y-R. The human P2Y2- R will be purified to homogeneity and reconstituted in proteoliposomes with G1 and other G proteins to: (i) unequivocally define agonist binding efficacies and potencies for the P2Y2-R, (ii) develop insight into the nature of constitutive activity of the P2Y2-R, (iii) determine the specificities of interaction of the P2Y2-R with G proteins, and (iv) establish whether agonist and G protein selectivities of the P2Y2-R are regulated by protein kinase-promoted phosphorylation or RGS proteins. Functional chimeric proteins will be constructed fusing the P2Y2-R with Galphaq, Galphai1, or Galpha12. Our preliminary results indicate that the human P2Y2-R, P2Y4-R, and P2Y6-R exhibit very different phenotypes of agonist-induced desensitization, internalization, and down-regulation. These differences in occurrence and characteristics of desensitization of P2Y-R may provide highly relevant to drug therapy of obstructive lung disease and other pathophysiologies. As such the molecular mechanisms underlying these differences will be elucidated. Our studies will include biochemical analyses that take advantage of expression of wild-type and dominant negative constructs of various regulatory proteins, e.g. G protein-coupled receptor kinases, beta-arrestins, and proteins involved in clathrin-related internalization, thought to be involved in agonist- dependent regulation of receptors. These studies will be complemented by studies that apply confocal microscopy to assess agonist-promoted translocation of P2Y-R and regulatory proteins in living cells. Key domains and residues involved in agonist-dependent regulation of the P2Y-R will be revealed by mutational analysis, and potential sites of phosphorylation will be confirmed by in vivo phosphorylation studies. The kinetics and regulation of agonist-dependent phosphorylation of P2Y-R by G protein receptor kinase 2 will be studied in proteoliposomes reconstituted with P2Y-R and Gq. This in vitro system will be utilized to directly define the mechanism(s) whereby P2Y-R phosphorylation regulates P2Y-R system will be utilized to directly define the mechanism(s) whereby P2Y-R phosphorylation regulates P2Y-R G protein coupling. Taken together these studies will provide novel molecular insight into P2Y-R biology and will generate heuristics for therapeutic strategies targeting P2Y-R in airway epithelial cells and other tissues.